KR20100087181A - Apparatus and method for electrochemical detection - Google Patents

Abstract

The present invention relates to a sensor with counter electrodes and a test strip using the sensor. The present invention can be used to measure blood or plasma coagulation in analytical assessments such as prothrombin time (PT) and thrombin potential, for example, in on-site care monitoring anticoagulants.

Description

Apparatus and method for electrochemical detection {APPARATUS AND METHOD FOR ELECTROCHEMICAL DETECTION}

The present invention relates to a sensor with counter electrodes and a test strip using the sensor. The present invention is directed to measuring blood or plasma coagulation in analytical assays such as prothrombin time (PT) and thrombin potential, for example in point-of-care monitoring anticoagulants. Can be used.

Various patents disclose devices and methods for detecting electrochemical substrates. For example, Jozefonvicz et al. (US Pat. No. 4,304,853) describe the use of electrochemical substrates to detect protease, including enzymes in a coagulation system. Unfortunately, these methods are not practical in field care equipment. The teachings, as used herein, are useful for the knowledge that the prerequisites of the electrode (s) are necessary to improve reproducibility because the measured current is very low. The substrate requires DMSO to aid in solubility in aqueous solutions. It also discloses a three-electrode system with a volume of sample and reagent that is much larger in digits than is required for practical field care testing.

Ludin et al. (US Pat. No. 6,495,336) typically disclose an electrochemical substrate that can be used for the detection of thrombin in a coagulation assay evaluation. The substrate provided herein has an improvement over that of Jozefonvicz et al. Because it is water soluble in appearance. They do not disclose how to use substrates in field care devices.

Frenkel et al. (US Pat. No. 6,352,630) disclose the use of electrochemical thrombin substrates, such as those disclosed by Ludin et al. (2002), in devices that appear to be suitable for on-site examination. The teachings as used herein provide that the substrate is fixed on the working electrode through an end opposite to the cleaved electroactive group. This requirement adds complexity and limits electrode materials and manufacturing techniques.

Immobilization of the substrate on the working electrode is important and / or necessary when using coplanar electrodes as taught by Frenkel et al. The reason is that the relatively large distance between the working electrode and the counter electrode prevents any meaningful diffusion of molecules from one electrode to another.

Likewise, the teachings above are preferred for coplanar electrode configurations because they provide Ag / AgCl reference electrodes, which may be counter electrodes, and because Ag / AgCl can act as a sink or source of electrons. A potential issue with this device is that in one embodiment it can only be detected if a relatively small amount of released electroactive product, which can be said to be in reduced form, is oxidized. This can result in a relatively low electrochemical current. In addition, the preparation of Ag / AgCl electrodes adds to the cost of manufacturing such sensors.

Unkrig et al. (US Patent Application Publication No. 2003/0146113 A1) also discloses the use of electrochemical thrombin substrates that are considered suitable for field care devices. As used herein, the reference describes a flat two-electrode system with a Pd working electrode and an Ag / AgCl coupled reference and counter electrode. In certain embodiments, to address the inherent low signal in this planar electrode design, the teachings relate to regenerating electroactive groups by combining their detection with oxidation of glucose. An advantage of this certain embodiment is that the counter electrodes can automatically regenerate the detected species without the need to bind to other redox reactions.

Opalsky et al. (European Patent Publication No. 1,234,053 B1) also describe a coagulation sensor using an electrochemical substrate. In certain embodiments, the teachings disclose a composite cartridge having a pump that preferably moves a sample in the cartridge to mix the sample plus reagent passing through the electrode in the same plane. In this regard, in certain embodiments herein, the counter electrodes allow simple strips that do not benefit from the pump as the reagents can be passively spread across the smaller volume of the sample.

A counter electrode biosensor is disclosed by Hodges et al. (US Pat. No. 6,284,125 B1) to measure the concentration of redox species. In particular, redox species were produced by oxidation of glucose from which glucose concentration was calculated. The counter electrode sensor may consist of the following:

(1) a working electrode and a counter electrode (and in some forms optionally a reference electrode) spaced apart by a predetermined distance;

(2) the electrodes oppose each other while lying on different but approximately parallel planes;

(3) Select the spacing between the working electrode and the counter electrode so that the reaction product from the counter electrode can diffuse into the working electrode.

While counter electrodes are disclosed by Newman and Chatelier (PCT / IB2007 / 001990) for coagulation sensors, this disclosure is limited to detecting coagulation using magnetic particle mobility (floatability) and teaches less about electrochemical substrates. Not.

Ohara et al. (US Pat. No. 6,620,310 B1) describe detecting a change in viscosity of a sample being solidified by detecting a decrease in steady state current through the counter electrodes. Again this disclosure does not relate to an electrochemical substrate, but instead a change in the diffusion of redox bonds is detected upon solidification.

None of the prior art describes the use of counter electrodes to detect an electrochemical substrate. They do not describe the unexpected finding that it is desirable not to fix the electrochemical substrate on the working electrode when using counter electrodes.

The present invention uses a sensor with counter electrodes and which can be used to measure blood or plasma coagulation in analytical assessments such as, for example, prothrombin time and thrombin potential in the field practice monitoring an anticoagulant. It is an object to provide a test strip.

Various embodiments of the present invention relate to an opposing-electrode sensor comprising a working electrode, a counter electrode and an electrochemical substrate, the sensor detecting degradation of the electrochemical substrate. The electrochemical substrate may be applied (coated) on the counter electrode. These sensors can be used as part of a test strip to test blood clotting time.

Another embodiment of the invention relates to a method of using the sensor and strip of the invention. These methods can be practiced in the field care.

1 includes a lower electrode 1, an insulating separator 2, an upper electrode 3, a cover 4 over a fill channel, a charge channel 5 and a reaction chamber 6. An exploded view of an exemplary electrochemical strip in accordance with an embodiment;
2 comprises a lower electrode 1, an insulating separator 2, an upper electrode 3, a cover 4 over a charging channel, a charging channel 5 and a reaction chamber 6 according to this embodiment. An exemplary assembled electrochemical strip;
3 shows an exemplary graph according to the present embodiment, which demonstrates improved clot detection when a thrombin substrate is applied on the state electrode; Time in seconds is given on the horizontal axis, and the current of the microamp is given on the vertical axis; Marked "a" in the graph indicates that the thrombin substrate is applied on the counter electrode (cathode in this example), and marked "b" in the graph indicates on the working electrode (anode in this example). The thrombin substrate is applied and thromboplastin is applied on the counter electrode (cathode).

Exemplary embodiments of the invention are disclosed in detail below. While certain exemplary embodiments have been disclosed, it is to be understood that this has been done for illustrative purposes only. Those skilled in the art will recognize that other components and forms may be used without departing from the spirit and scope of the invention. The expression “one embodiment”, “one embodiment”, “exemplary embodiment”, “various embodiments”, etc. includes embodiments, such that the described embodiment (s) includes particular characteristics, structures, or features. Although it may be, it may mean that all embodiments do not necessarily include such special features, structures or features. Also, repeated use of phrases such as "in one embodiment" or "in an exemplary embodiment" does not necessarily refer to the same embodiment, but they may refer to the same embodiment.

The present invention relates to a sensor having opposing electrodes (eg, working electrode and counter electrode) and an electrochemical substrate. In some embodiments, the electrochemical substrate is on the counter electrode. As described below, these sensors can be used by applying a sample to the sensor (optionally on a test strip), where the protease degrades the electroactive species of the substrate. These electroactive species can be detected by redox reactions at the electrodes.

The present invention is in a wide range of techniques, for example, in methods for measuring blood or plasma coagulation using analytical assessments such as prothrombin time (PT) and thrombin potential, in particular, oral anticoagulants (eg, warfarin and phenprocumon ( phenprocoumon) can be used in on-site medical monitoring.

As will be described further below, one advantage of the present invention is that the counter electrodes of certain embodiments do not require the complexity of a true reference electrode such as an Ag / AgCl electrode or the like. Instead, in these embodiments, the tightness of the electrodes allows for cycling of the electroactive species. That is, molecules reduced at the cathode may be reoxidized at the anode, resulting in higher currents. This may make it possible to use two similar electrodes, such as those formed of inert conductive materials such as carbon, Au, Pd, Pt Ir, indium oxide, mixed indium / tin oxide and / or tin oxide and the like.

The device and test strip of the present invention have an electrochemical substrate that can be dried on the counter electrode in some embodiments. Electrochemical substrates can be degraded by enzymes such as proteases. Proteases are a class of enzymes that break down peptide bonds. They play an important role in many biological processes. For example, they are involved in food digestion, HIV replication, blood clotting cascades and alternative pathways.

Some proteases are specific for certain peptide sequences, while others are less selective. In either case, it may be highly desirable to detect and / or quantify protease activity as this provides insight into biological processes. As will be appreciated by those skilled in the art, any natural or genetically engineered protease or other degradation enzyme may be detected, or in other cases, the enzyme recognizes and degrades the desired bond or bonds in the electrochemical substrate in use. It can be used in the present invention under the condition that it is possible to.

Many useful peptide substrates are commercially available for a range of proteases. Typically, proteases degrade dyes or fluorescent groups from these substrates, resulting in a change in color or fluorescence. It is also possible to create substrates with electroactive groups that can be detected electrochemically after decomposition. The amino acid sequence of the peptide substrate determines the specificity for the protease. As such, in some embodiments, the specificity of a protease detection method can be varied by the selection of a substrate specific for the protease of interest.

One exemplary protease detection system that an embodiment of the invention can detect is a blood coagulation cascade. This blood coagulation cascade consists of a series of protease reactions, which serve as a model system for investigating the detection of proteases, depending on their practical use. The time it takes to coagulate a sample of blood or plasma can indicate various disease states, which can be useful for monitoring anticoagulation therapy. The specificity of such coagulation tests is usually not due to how coagulation is detected, but due to the nature of the reagent (s) used to initiate coagulation. Thus, this embodiment is applicable to a wide range of clotting tests.

For example, one of the fields of use for the present invention is a method of monitoring anticoagulation by oral vitamin K antagonists such as warfarin and fenprocumon in field care. These drugs are generally monitored using a prothrombin time test and the results are reported as international normalized ratio (INR). These tests tend to be performed using field devices that provide results in minutes with blood derived from simple finger-pricks. This approach is usually much more convenient than traditional venipuncture and plasma preparation, but such devices require a method for detecting whole blood clotting. This embodiment improves the technique which copes with this point.

Plasma coagulation can be detected in a variety of ways. For example, gelation of a sample can be detected by increased resistance to particle (typically magnetic) migration through the sample or by a change in the fluidity of the sample. The gel point can also correspond to the increasing turbidity of the plasma, which can be detected optically. Electrochemical methods are described, for example, in US Pat. No. 6,673,622; 6,066,504; 6,066,504; 6,060,323; 6,060,323; And 6,046,051, which can be used to detect a change in the viscosity of a sample by measuring a change in impedance.

Coagulation may be detected indirectly using an artificial peptide substrate for thrombin, depending on the embodiment. For example, colored substrates can be degraded by thrombin (or other coagulation enzymes), and dye groups can be dissociated. This causes a detectable color change in solution. Other substrates may be provided if the degraded group is electrochemically active (can be oxidized and / or reduced at the electrode) and can be detected at the electrochemical sensor. This type of substrate is referred to as an electrochemical substrate, which is the subject of certain embodiments herein.

For the purposes of this embodiment, an electrochemical substrate is defined as a chemical compound that is degraded by an enzyme to dissociate an electroactive leaving group. The native substrate has less or different electrochemical activity, under detection conditions, as compared to free leaving groups dissociated by degradation.

Electroactive species can be detected by current measurement in embodiments of the invention, for example using a two or three electrode system. The three-electrode system can have a working electrode, a counter electrode and a reference electrode. The reference electrode measures the voltage of the solution near the surface of the working electrode. The potential between the working electrode and the counter electrode can be adjusted such that the voltage between the working electrode and the counter electrode is set to a desired value. The magnitude of the current through the circuit connecting the working electrode and the counter electrode is determined by the rate of reduction or oxidation at the solution interface of the working electrode. In this way, the place where the target reaction occurs is the working electrode. The system is designed such that the reaction at the working electrode rather than at the counter electrode limits the current.

In one embodiment, the two-electrode system does not have a separate reference electrode. Instead, the voltage between the conductors of the other two electrodes can be set. The working electrode may again be defined as the electrode in which the target reaction occurs. This will be the electrode where the current limiting reaction occurs.

The inventors have surprisingly found that electrochemical sensors with counter electrodes can have a number of advantages over using electrodes in the same plane, including but not limited to:

(1) Small electroactive species can easily diffuse from one counter electrode to another. This may be due to the recycling of the reduced and oxidized forms, effectively amplifying the signal. This is not possible with electrodes in the same plane, because the distance is so large that the species do not spread the necessary distance to the working electrode usefully in time.

(2) As will be appreciated by those skilled in the art, any redox reaction needs to have an oxidizing component and a oxidizing component. In the counter electrode sensor, the subject electroactive species can be oxidized at one electrode and reduced at the other electrode in the aforementioned recycling. In electrode sensors in the same plane, extra effort is required to ensure that a reaction at the counter electrode can occur. For example, the counter electrode may be Ag / AgCl, or a large voltage may be applied, or complementary electroactive species may be required.

(3) The close contact of the opposite electrodes to each other means that most of the potential difference between the electrodes is occupied by the voltage on the electrode interface, not on the volume of the solution. In this regard, electrodes in the same plane may have a significant I × R voltage drop over the sample, especially if the solution between the electrodes has a low ionic strength in one embodiment.

(4) Since the manufacture of the counter electrodes may require the deposition or removal of a metal (or other conductive material) to form separate electrodes that are electrically insulated from each other in forming electrodes in the same plane, It may be easier than the electrodes in the plane. In this regard, it may be advantageous to have a conductive surface covering the entire material forming each counter electrode. This means that the electrode is applied using a simple process such as, for example, a sputtering coating and no specific pattern in the conductor is required. The counter electrode region that the sample contacts may be defined by an insulating spacer separating the electrodes.

(5) The use of counter electrodes can avoid the requirement in certain embodiments of Frenkel et al. (US Pat. No. 6,352,630; 2002), which requires the immobilization of an electrochemical substrate in a particular orientation. In the counter electrode sensor, the electrochemical substrate can be released and dissolved in the sample, the orientation of which is irrelevant. This simplifies the manufacturing process.

One or more embodiments relate to a strip shaped device (ie, an instrument) for measuring protease activity, for example to monitor blood coagulation. The strip is electrically connected to a measuring device that measures the current or voltage obtained by applying a predetermined voltage or current to the strip. As will be appreciated by those skilled in the art, these methods are each known, for example, as current measurement or galvanostatic electrochemical techniques. The meter can also thermally control the strip and calculate, record, display and / or transmit the results of the reaction.

The exemplary strips shown in FIGS. 1 and 2 may comprise two electrodes 1, 3 in opposition. These electrodes can be electrically insulated from each other by the plastic separator 2 or an equivalent thereof. In some embodiments, these electrodes may be 0.5 mm or less, in certain embodiments 0.05-0.15 mm apart.

The separator 2 can have a gap therein defining the reaction chamber 6 by forming a cavity between the electrodes. The electrode surface is electrically conductive, in electrical contact with the meter.

In certain embodiments, the surface is sputtered coated on a plastic support. Suitable coatings may be known to those skilled in the art and include, for example, platinum, indium, carbon, mixed indium / tin oxides, and tin oxides, but are preferably palladium or gold.

The surface on each electrode may be the same or different. The conductive surfaces face each other. The filling channel 5 allows a sample to be introduced into the reaction chamber, while the reaction chamber can be maintained at a set temperature in the meter.

Drying reagent (s) that can be used to detect protease activity can be applied on the surface of the reaction chamber. In certain embodiments, the electrochemical substrate is not applied on the working electrode. After addition of a sample (eg blood), degradation of the substrate dissociates the electroactive species, resulting in a detectable change in electrochemical activity. The reagent (s) may also contain components that activate or facilitate blood or plasma coagulation enzymes.

In certain embodiments, the disclosed strip consists of two electrodes of opposite types. These electrodes may comprise palladium sputter coated on a plastic support. A 0.1 mm thick insulating separator layer may define separation of the electrodes. One or more gaps in the separator may define the reaction chamber (s). The filling channel can allow the sample to be transferred into the reaction chamber (s) by capillary action.

Drying reagent (s), which may be useful and / or necessary for initiation or detection of coagulation, may be applied on the surface of the reaction chamber (s). These reagents can be, for example, contact activators, tissue factors, animal poisons, phospholipids, buffers, preservatives, stabilizers, dyes, surfactants and combinations thereof. These dry reagents can react when a blood or plasma sample is introduced into the reaction chamber. Activated coagulation enzymes degrade the electroactive groups from the substrate present in the reagent (s).

In specific embodiments, the reagent may contain a compound, including thromboplastin, required for prothrombin time testing.

In a further embodiment, the reagent may contain a compound such as dilute thromboplastin or tissue factor, which is necessary for thrombin generation test, in which parameters such as delay time, thrombin peak and endogenous thrombin potential can be derived.

In another embodiment, the reagent may contain any compound containing an activator from the poison, which is necessary for testing, such as the dilute Russel viper venom test or ecarin clotting time test of Russell. .

In other embodiments, the reagent may contain factor Xa and / or thrombin and / or antithrombin to determine factor Xa or anticoagulant that inhibits thrombin activity.

In other embodiments, the reagent may contain plasma components (eg, from bovine plasma) to compensate for variations in coagulation factors such as factor V and fibrinogen. This may be similar to the Owren PT test.

In some embodiments, it is advantageous to apply one or more electrodes with an electroactive compound that ensures that the counter electrode does not limit the current. For example, if the cathode is a counter electrode, applying the electrode with iron (III) EDTA provides a reducing compound that supports the detection of the target species (in this embodiment, by oxidation). This may be particularly advantageous if the species of interest is not fully electrochemically reversible.

In some embodiments, it has proved advantageous to dry the thromboplastin and electrochemical thrombin substrates on the counter electrodes to prevent back reaction between the two reagents. In one exemplary embodiment, the electrochemical thrombin substrate (toluolsulfonyl-glycyl-prolinyl-arginine-4-amido-2-chlorophenol) can be applied on the counter electrode and the trombo Platinum may be applied on the working electrode. In this example, the leaving group of the thrombin substrate is broken down into the reduced form. Here, this is the species to be detected and limits the current. Since the leaving group is in reduced form, it is oxidized on the anode to pass a current, whereby the anode is the working electrode.

In general, applying the thrombin substrate on the working electrode was expected to result in faster clot detection because the leaving group dissociates in the detected electrode. However, in some embodiments, the opposite applies. For example, FIG. 3 shows that the thrombin substrate is faster when dried on the counter electrode (cathode) rather than the working electrode (anode). The faster reaction rates of these embodiments make it possible to determine the clotting time more consistently.

Many biological assay evaluations (eg, thrombin generation assay evaluations) include calculating reaction rates. In certain embodiments, it is observed that when chemically generated electroactive species diffuse into the working electrode rather than occur in the working electrode, it may be easy to measure the reaction rate of those species. This may be referred to, for example, in US Pat. Nos. 5,942,102 and 6,444,115. This is another reason that in certain embodiments it may be advantageous to apply the electrochemical substrate away from the working electrode.

In other embodiments, the electrochemical substrate and other reagents (eg, thromboplastin) can be formulated so as not to interact in reverse, allowing all or reagent components to be applied together on the counter electrode. . This has the advantage of keeping the working electrode in the absence of any reagents that may contaminate the working electrode. Contamination is not a problem for the counter electrode because the counter electrode does not limit the current. Contamination of the working electrode may not be immediately apparent, and sometimes only after preservation, which may be a cause of shortening of life for the product embodying the present invention. In this way, application on the counter electrode can avoid the risk of contaminating the working electrode.

The test strip of the present invention may also include means for verifying the strip and / or sample integrity. In one embodiment, the strip has a reaction chamber containing reagents that function to verify strip and / or sample integrity. The reagent may in other cases include a coagulation factor lacking in the sample. This reaction chamber can exhibit approximately normal solidification time regardless of the lack in the sample and can be used to demonstrate strip integrity if the solidification time is within a predetermined limit.

In other embodiments, strip integrity can be verified by including an electroactive compound in the reagent. Such compounds may differ from electrochemical substrates, which may possibly only dissociate the electroactive groups upon enzymatic degradation. The compounds that monitor strip integrity can change (increase or decrease) electrochemical activity under preservation conditions (eg, elevated temperature or humidity) that may damage other components of the strip. Examples of such compounds include 4-amino-2-chlorophenol, ferricyanide, ferrocyanide, and organic N-oxides or nitrozo compounds disclosed in US Patent Application Publication No. 2005/0123441. The electrochemical activity of these compounds can be contrasted with predetermined thresholds for batches of strips. If the value is outside the threshold, the meter may give an error message instead of the result.

Reagents for monitoring strip integrity may be in the same reaction chamber that detects substrate degradation, or they may be in different chambers. In embodiments where the chambers are separate, these chambers may be electrically insulated by the discontinuity of the electrodes, or in alternative embodiments, these chambers may use electrically connected electrodes. If the chambers share continuous electrodes, the signal detected by the meter may be a combination of chambers. Interference between the integrity and degradation reaction signal can be minimized by detecting the integrity reaction before and after the decomposition reaction and / or by reducing the cross reaction electrode region.

Some embodiments of the present invention relate to on-board control suitable for monitoring the viability of electrochemical biosensor strips, which is not limited to those disclosed herein. The electrode materials and the application on them are employed such that a potential difference is established between the electrodes. This voltage can be measured directly or the current causing it is measured. In some embodiments, the sensor behaves like a battery that simply generates a charge. The potential difference collapses after a relatively short time and does not interfere with subsequent test reactions.

Such control is useful, for example, in field care testing, based on a meter that measures the electrochemical response in the most commonly used disposable strips. There is a need to ensure that the test results are accurate and control to ensure strip integrity is desired. For the end user, eg a patient, the most convenient control is to verify the integrity of the same strip under test with an "on-board" strip. An example of on-board control can already be found in the field of on-site coagulation device (eg Coaguchek XS, INRatio). In addition, US Patent Application Publication No. 2005123441 A1 provides a concise introduction to the benefits of on-board.

Electrochemical biosensors typically become more complex in further separating the control reaction from the test reaction, for example, increasing complexity:

a. Strips with a single reaction chamber containing both control and test reactions are relatively easy to manufacture.

b. Strips with separate but electrically connected control chambers and test chambers become more complex as they require the provision of multiple reagents in different regions.

c. Strips with separate and electrically insulated control chambers and test chambers can be a significant challenge for manufacturing on an economic scale. This usually requires the deposition or removal of the electrode material in a predetermined pattern.

However, the difficulty in finding an appropriate controlled reaction chemistry is the reverse of that discussed above:

a. Strips with separate and electrically insulated control chambers and test chambers will not have any problem where one reaction (eg, control) interferes with another reaction (eg, test).

b. Strips with separate but electrically connected control chambers and test chambers may be subject to electrical interference between the two chambers since electrons from one reaction cannot be distinguished from electrons from another reaction, but control and test Will not interfere chemically.

c. Strips with a single reaction chamber require components that do not chemically interfere with each other or cause interfering electrical signals.

The present invention, in some embodiments, relates to a novel way of imparting a control response in an electrochemical sensor as well as a method of preventing the electrical signal from the control reaction and the electrical signal from the test reaction from interfering with each other.

US Patent Application Publication No. 2005123441 A1 discloses an on-board controlled reaction using N-oxides or nitrozo compounds that are reduced when exposed to conditions that may impair strip performance. Changes in on-board control can be detected optically or electrochemically. The electrochemical detection method disclosed in US Patent Application Publication No. 2005123441 A1 involves applying -700 mV to Ag / AgCl across the working electrode and then applying -100 mV.

Embodiments of the present invention may be advantageous because the control of the present invention requires only 1-2 seconds instead of being 4.5 seconds in some existing devices. The control of the present invention is free from the risk that the applied voltage will interfere with the test.

One specific embodiment includes on-board control to monitor the viability of an electrochemical sensor that detects blood coagulation. In this specific embodiment, the control reaction reagent measuring the feasibility of the strip is contained in the same chamber as the test reaction reagent measuring the clotting time. The signals from the control response are differentiated from the signals of the test by the time they occur. Specifically, the control reaction is imparted within the first few seconds after adding the sample, followed by the test reaction.

In this embodiment, the control reaction may be made with a low (eg 0.5 mM) ferricyanide concentration applied on one electrode and free of ferricyanide on the other electrode. The difference in electrode chemistry creates a voltage across the electrode when the sample is added. This voltage can be measured directly by a voltmeter, or in another preferred embodiment, the current through an external circuit can be recorded by an ammeter.

The electrochemical voltage can be dissipated by at least two methods: the current flowing through the external circuit effectively flattens the electrochemical battery, and the diffusion of the ferricyanide from one electrode to the other leads to The concentration gradient of the cargo, thus reducing the voltage. Once the signal from the control reaction is dissipated, analysis of the test response can begin. This can consist, for example, of applying 0.3 to 0.4 V across the electrode and measuring the resulting current measurement current.

In some embodiments, the concentration of ferricyanide is considerably low such that it is substantially uninterrupted by the detection of the test response. However, as will be appreciated by those skilled in the art, other embodiments may be made using alternatives to ferricyanide. For example, iodine, ascorbate, ferrocyanide, 4-amino-2-chlorophenol can have a battery effect.

In one embodiment, the battery effect is measured by measuring the current without applying any external voltage across the sensor electrode. However, it is possible to measure the current by applying a small voltage under the condition that the voltage does not cause substantial interference with the test response.

Embodiments of the present invention may include having a sensor for receiving two chambers. One chamber may contain test-reaction reagents and the other chamber may contain control-reaction reagents. This approach is advantageous if the components of one reaction (eg a control reaction) can chemically inhibit the other reaction (eg a test reaction). These chambers need not be electrically isolated under the condition that the test and control reactions do not electrically interfere with each other. This embodiment is particularly advantageous if a neutralizing agent is included in the control reaction chamber (eg on the counter electrode).

In other embodiments, concentrations of control-reactant reagents that would normally interfere with the test-reaction may be used. In this embodiment, the control chamber contains separate regions of control reagent and neutralizer. When the sample is added, the control reagent produces a battery effect or some other electrochemical signal but it is quickly neutralized by the neutralizer. Neutralization may be due to a number of physical processes, such as chemical reactions (e.g., search and neutralize each other iodine ascorbate) or precipitation (e.g., Co ^{+ 2} or Mn ^{+ 2} ions having to precipitate a ferry cyanide). Precipitation of the electroactive compound may make it impossible to interact with the electrode.

In one embodiment for measuring blood or plasma coagulation, the meter can maintain a sensor at 37 ° C. The electrical signal detected by the meter can be interpolated to calculate the clotting time. For example, the clotting time may be defined as the point at which the signal or its rate of change reaches a threshold. The clot time may be recorded, displayed or otherwise output in units of time, or may be converted to different units such as INR using calibration data assigned to sensors and / or meters.

Other results may also be calculated and reported. For example, according to the correct reagent, the area under the curve may indicate endogenous thrombin potential, which may be reported as well. The analysis evaluation results may be reported by being displayed on the screen of the meter, but this may be stored in memory and / or transmitted to another device.

Embodiments of the present invention also relate to kits having the sensors or test strips described herein. Such kits may be sterile packaged. Test strips or sensors can be provided in individual packaging as well as in multiple packages. Optionally the kit includes a meter for interacting with the test strip or sensor and / or instructions for using the kit, test strip or sensor.

All publications cited herein, including websites, journal articles or abstracts, publications or publications of corresponding US or other foreign patent applications, or other publications, shall contain all data, tables, drawings, and text presented in that publication. The entirety of which is incorporated herein by reference. For example, the following documents are specifically incorporated by reference in their entirety, if any, including their related commonly invented and / or assigned references: US Pat. No. 4,304,853 (inventor: Jozefonvicz et al.); US Patent No. 5,942,102, published by Hodges et al .; US Patent No. 6,060,323 published by Jina; US Patent No. 6,066,504 (Jina); US Patent No. 6,046,051 published by Jina; US Patent No. 6,248,125 (inventor: Hodges et al.); US Patent No. 6,444,115, published by Hodges et al .; US Patent No. 6,352,630 to Frankel et al .; US Patent No. 6,495, 336 (inventor: Ludin et al.); US Patent No. 6,620,310 (Ohara et al.); US Patent No. 6,673,622, published by Jina; US Patent Application Publication No. 2005/0123441 (Inventor: Unkrig et al.); US Patent Application Publication No. 2003/0146113, published by Unkrig et al .; European Patent 1,234,053 (inventor: Opalsky et al.); International Patent Application No. PCT / IB2007 / 01990 (Inventor: Newman et al.); Neurath, H. and Walsh, K.A. (1976), Role of proteolytic enzymes in biological regulation (a review), Proc. Natl. Acad. Sci. USA. 73: 3825-3832; Spronk, H.M.H. Et al. (2003), The blood coagulation system as a molecular machine, BioEssays, 25: 1220-1228; And Bates, S. M. and Weitz, J. I. (2005), Coagulation assays. Circulation. 112: 53-60.

While the invention has been particularly shown and described with reference to some embodiments thereof, those skilled in the art have been presented by way of example only and are not intended to be limiting, and various changes in form and detail may be made without departing from the spirit and scope of the invention. You will understand that. Accordingly, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents. Any headings used herein are provided for organizational purposes only and are not intended to impart any boundaries or meaning to this specification except where specifically noted.

1: lower electrode 2: insulated separator
3: upper electrode 4: cover over charging channel
5: filling channel 6: reaction chamber

Claims (19)

An opposing-electrode sensor for detecting cleavage of the electrochemical substrate, including a working electrode, a counter electrode and an electrochemical substrate. The counter-electrode sensor of claim 1, wherein the electrochemical substrate is not fixed on the working electrode. The counter-electrode sensor of claim 1, wherein the electrochemical substrate is a substrate for thrombin comprising a peptide bound to a leaving group that is once decomposed by thrombin and becomes electroactive. The counter-electrode sensor of claim 1, further comprising a meter connected to the sensor, wherein the meter determines a solidification time for the sample under test. The counter-electrode sensor of claim 4, wherein the measuring device displays or outputs a result including a solidification time or a result or value depending on the solidification time. The counter-electrode sensor of claim 1, further comprising a meter connected to the sensor, the meter displaying or outputting a result including a parameter of thrombin generation in the sample tested. A test strip for measuring blood or plasma coagulation time in a sample comprising a counter-electrode sensor comprising a working electrode, a counter electrode and an electrochemical substrate, wherein the sensor detects degradation of the electrochemical substrate. . 8. The test strip of claim 7, wherein the electrochemical substrate in the sensor is not immobilized on the working electrode. 8. The test strip of claim 7, wherein said substrate in said sensor is a substrate for thrombin comprising a peptide bound to a leaving group that becomes electroactive once degraded by thrombin. 8. The test strip of claim 7, further comprising a meter connected to the sensor, the meter determining the solidification time for the sample under test. The test strip of claim 10, wherein the meter displays or outputs a result including a solidification time or a result or value depending on the solidification time. 8. The test strip of claim 7, further comprising a meter connected to the sensor, the meter displaying or outputting a result comprising a parameter of thrombin generation in the sample tested. 8. The test strip of claim 7, further comprising means for verifying strip integrity. A method of measuring blood or plasma coagulation,
Obtaining a blood sample;
Applying the blood sample to a counter-electrode sensor comprising a working electrode, a counter electrode and an electrochemical substrate; And
Analyzing the sample using the sensor to determine at least one blood or plasma coagulation property of the sample,
Wherein said sensor detects degradation of said electrochemical substrate. The method of claim 14, wherein the method is performed at point-of-care. The method of claim 14, further comprising connecting a meter to the sensor, wherein the meter displays or outputs a result including a parameter of thrombin generation in the tested sample. . The method for measuring blood or plasma coagulation according to claim 14, wherein the patient is taking an anticoagulant. 18. The method of claim 17, wherein the anticoagulant is a vitamin K antagonist. The method of claim 14, wherein the sample is obtained from the patient by pricking the patient's finger.

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